This project proposes the continuation of studies on the molecular pathology of human hereditary diseases which are characterized by defects in the cellular processing of damaged DNA, and their relationship to cancer. The recent observation that the human ERCC2 gene corrects the repair-defective phenotype of cells from individuals with xeroderma pigmentosum (XP) complementation group D, will be investigated further. XP-D cells, including cells from XP-D individuals with the associated disease trichothiodystrophy (TTD), will be investigated for the presence of mutations in XP-D alleles and mutations will be mapped and characterized. The cloned ERCC2 gene will be overexpressed and its protein product will be purified and characterized in detail. Gene replacement experiments will be carried out in mouse embryonic stem (ES) cells by homologous recombination with the genomic mutated mouse ERCC2 gene. These cells will be injected into blastocysts which will be brought to gestational maturity in pseudopregnant mice. Recombinant heterozygous mice will be bred to generate homozygous mutant strains carrying defects in the ERCC2 gene. The mice will be used to study the pathobiology of defective DNA repair under controlled environments and in the presence of known carcinogens. A recently developed Epstein Barr virus (EBV)-based episomal cloning vector will be used to screen human cDNA libraries for genes which complement the UV sensitivity of XP cells from genetic complementation groups C, F, G and V(variant), as well as cells from Cockayne's syndrome (CS) from genetic complementation group A.